The metalloproteinase gene family, ADAM (a disintegrin and metalloproteinase), includes members that are membrane-anchored proteases with diverse functions. ADAMTS family members are distinguished from ADAMs by the presence of one or more thrombospondin 1-like (TSP1) domain(s) at the C-terminus and the absence of the EGF repeat, transmembrane domain and cytoplasmic tail typically observed in ADAM metalloproteinases.
A Disintegrin-Like and Metallopeptidase with Thrombospondin Type 1 Motif 13 (ADAMTS13) is a member of the ADAMTS family. ADAMTS13 has eight thrombospondin domains and no hydrophobic transmembrane domain. Accordingly, it is secreted. ADAMTS13 cleaves von Willebrand Factor at the Tyr1605-Met1606 bond and requires both calcium and zinc ions to function. ADAMTS13 is also known as “von Willebrand Factor-Cleaving Protease” and “VWFCP.”
Deficient ADAMTS13 expression has been implicated in the pathogenesis of some diseases, e.g., thrombotic disorders such as thrombotic thrombocytopenic purpura (TTP) (see, e.g., U.S. Patent Publication No. 20070015703). In TTP, deficiency and/or inhibition of ADAMTS13 results in extensive microscopic thomboses that form in small blood vessels throughout the body (thrombotic microangiopathy). Red blood cells passing through the microscopic clots experience shear stress, which causes damage to the red blood cell membrane, and which in turn leads to intravascular hemolysis and schistocyte formation. Thromboses also cause reduced blood flow, which can result in end organ damage. Symptoms typically include neurological problems, such as hallucination, bizarre behavior, altered mental status, stroke, or headaches; kidney failure; fever; and thrombocytopenia (low platelet count), resulting in bruising or purpura; and microangiopathic hemolytic anemia, involving anemia and jaundice. Current therapy involves plasmapheresis to reduce circulating antibodies against ADAMTS13, and/or replenishing blood levels of the enzyme.
Therefore, a strong need exists for providing methods of purifying recombinant ADAMTS13, particularly on a commercial production scale, which may be used as a therapeutic agent. Purification of ADAMTS13 has proven difficult and various approaches have been attempted, including chromatography. A chromatographic material that binds non-ADAMTS13 protein, allowing the ADAMTS13 protein to appear in the eluate or supernatant, would provide a useful approach for purification. A chromatography material that binds ADAMTS13 protein, while non-ADAMTS13 impurities either remain in solution or bind much more strongly, also presents an attractive approach, and may be used in tandem with other approaches. The instant disclosure provides such approaches.
Furthermore, virus contaminants have posed additional challenges in the purification of ADAMTS13 proteins, as well as other proteins and recombinant proteins. One conventional approach involved treating a sample to be purified with a solvent-detergent mixture in solution. Incubation of the sample with the solvent-detergent chemicals led to deactivation of lipid-coated viruses. This in-solution treatment, however, inefficiently required transfer of the sample to at least one other vessel, e.g., to facilitate removal of the solvent-detergent chemicals after treatment. Further, some proteins including ADAMTS13 are sensitive to the solvent-detergent chemicals, resulting in aggregate formation. The instant disclosure provides an approach involving immobilization of the protein during the solvent-detergent treatment to address such problems of virus inactivation.